Telomeres are regions of repetitive sequence located at the ends of DNA strands that inhibit deterioration of the vulnerable DNA ends and prevent fusion with neighboring DNA. They provide a buffer to the vital genes located near the ends of chromosomes as DNA at these ends are consumed with rounds of cellular division. In almost all species that have cells with linear chromosomes, telomeres consist of G-rich repeats and associated proteins. In human cells, telomeres are comprised of between less than 0.5 kb to more than 20 kb of (TTAGGG)n repeats which are in dynamic equilibrium with a specific set of proteins. The length of telomeres is heterogeneous but telomeres are present consistently at the end of each chromosome (Moyzis, R et al. (1988) Proc Natl Acad Sci. 85: 6622-6626).
The shortening of telomeres over time causes cells to reach a limit in their replication and eventually progress to senescence (Blackburn, E. (2000) Nature 408(6808):53-56). Telomere shortening protects against tumorigenesis by limiting cell growth (3, 4), but also can impair tissue regenerative capability and cell viability (5, 6). Shortening of the telomeres can also lead to the activation of a DNA damage response and DNA repair pathways. In some instances, cells are immortalized via telomere lengthening or the activation of telomerase, a telomere lengthening enzyme (Eisenberg, D. (2011). American Journal of Human Biology 23 (2): 149-167; Chang, S et al. (2003) Genes Dev; 17:88-100). This Alternative Lengthening of Telomeres (ALT) results in genomic instability and leads to disorders, including cancer and age-related diseases. This gradual loss of telomere repeats contributes to replicative senescence or apoptosis in human cells was confirmed and loss of telomeres has been implicated in genomic instability and neoplastic transformation as well as many age-related diseases including aplastic anemia, pulmonary fibrosis or cancer (Murnane et al. (1994). EMBO J. 13: 4953-4962.). Hence, the understanding of these complex disorders relies on the study of telomeres and the ability to measure the lengths of telomeric repeats in cells.
The realization that the length of telomere repeats at individual chromosome ends is a critical variable in cell fate decisions and biological functions ranging from aging to carcinogenesis has highlighted the need for techniques that can provide accurate information on the length of telomeres in different cell types.
A number of techniques currently exist in the art which aim to measure telomere length. Reviewed, e.g., in Aubert et al. (2012) Mutat. Res. 730:59-67. Thus far, most assays of telomere length measure average telomere length from aggregates of many cells derived from dissected tissues, cultured cells or blood (7).
Terminal Restriction Fragment (TRF) (8) utilizes Southern blot analysis to measure the telomere length of a population of cells to determine the average telomere length of the population. Restriction enzymes which specifically exclude telomere repeats are employed to digest genomic DNA to yield short genomic fragments and longer uncut telomeres. The telomere fragments are separated by agarose gel electrophoresis and detected through either Southern blotting or a hybridization technique which uses a labeled probe to detect telomeric DNA (Kimura, M. (2010) Natl Protoc. 5: 1596-1607). This is used to estimate average genomic telomere length by comparison to a DNA ladder size standard and normalization to a reference sample. Attaining sufficient results using TRF requires large amounts of DNA (0.5-5 μg) and several days for processing. Moreover, the requirements for gel electrophoresis and hybridization limit the scalability of this assay. Further, TRF data cannot be readily comparable across studies because techniques are not standardized with respect to restriction enzyme selection, starting DNA quantity and quality, and blot analysis. The technique is also not sensitive enough for short telomeric length measurements.
Single TElomere Length Analysis (STELA) involves the application of single molecule PCR to generate telomere measurements from limited starting material (20 cells or more). The method involves annealing a linker to the telomere overhang and subsequently introducing a linker-specific primer and a primer specific for a unique subtelomeric sequence in a small-pool PCR reaction to generate an individual amplicon for each single telomere (Britt-Compton, B. (2006) Hum Mol Genet. 15: 725-733). The biggest drawback of STELA is its requirement for suitable sequences at the ends of the chromosome, and hence, it is only appropriate for several well-characterized ends.
QPCR and related method of MMQPCR (monochrome multiplex QPCR) amplify C- and G-rich strands of the telomere using primers with mismatches present to avoid primer dimer formation and to ensure amplification of solely the telomeric region. This amplification is quantitated and correlated to that of a single copy gene (Cawthon, R. (2002) Nucleic Acids Res. 30:e47; Cawthon, R. (2009) Nucleic Acids Res. 37:e21; U.S. Patent Appl. Publ. No. 2011/0294676). While the DNA requirement (35 ng or more) for QPCR is significantly less than TRF, it still relies on populations of cells to derive sufficient amounts of DNA.
Quantitative fluorescence in-situ hybridization (QFISH) allows sensitive visualization of relative telomere length from individual cells and individual telomeres, but this method requires many cells and/or metaphase arrested cells, which precludes its application to many sample types, including post-mitotic cells, senescent cells and other non-dividing cells, and when only one actual cell is required to test. In addition, preparing chromosome spreads requires significant technical skill, and only proliferating cells within a population reach metaphase stage, so this analysis potentially biases the estimates of telomere length for a given cell population (10-12). High-throughput quantitative FISH (HT QFISH), Flow FISH and STELA can be used for telomere measurement of dividing, non-dividing and senescent cells, but these methods also require large cell populations (13-15).
Each of the above assays, in addition to their individual drawbacks, cannot be effectively utilized to measure telomere length in single cells in high-throughput format.